In situ dilution of external controls for use in microarrays

ABSTRACT

An array in which an external control feature for normalization has been designed and tested for its ability to mimic the range of observed expression levels for a test set of oligos. The external control probes span a series of concentrations. They are spatially randomized across a grid of an array. The series of concentrations is duplicated in a given grid. The individual grid layout and number of control and external normalization features per grid have been designed to cope with sources of both systematic error and spatial variation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of a U.S. Provisional PatentApplication No. 60/619,008, filed Oct. 18, 2004, the entire disclosureof which is hereby expressly incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is related to the field of arrays morespecifically to array systems.

2. Description of the Related Art

DNA arrays are commonly used to make quantitative or relativemeasurements of gene expression. They provide a medium for matchingknown and unknown DNA samples based on base-pairing rules and automatingthe process of identifying the unknowns. In general, arrays aredescribed as macroarrays or microarrays, the difference being the sizeof the sample spots. Macroarrays contain sample spot sizes of about 300microns or larger and can be easily imaged by existing gel and blotscanners. The sample spot sizes in microarrays are typically less than200 microns in diameter and these arrays usually contain thousands ofspots.

The microarrays contain nucleotide sequences corresponding to knowngenes or expressed sequence tags. A single microarray can containthousands of genes, which may represent a significant subset of thegenes, or even the entire genome, of an organism. A comparison of cellsor tissues from experimental and control preparations provides data ondifferences in expression levels between the two conditions. For thispurpose, mRNA is extracted from a sample, converted to complementary DNA(cDNA) and tagged with a fluorescent label. In a typical microarrayexperiment, cDNA from one sample (sample A) is labeled with a first dyethat fluoresces in the red and cDNA from another sample (sample B) islabeled with a different dye that fluoresces in the green. Thefluorescent red and green cDNA samples are then applied to a microarraythat contains DNA fragments (oligonucleotides) corresponding tothousands of genes. If a DNA sequence probe is present on the microarrayand its target complement is present in one or both samples, thesequences bind, and a fluorescent signal can be detected at the specificspot on the array. The signals are generally picked up using a “scanner”which creates a digital image of the array. The red to greenfluorescence ratio in each spot reflects the relative expression of agiven gene in the samples A and B.

Current microarray analyses rely on normalization and quality controlmethods that often assume evenly distributed changes, and/or absence ofglobal shifts in gene expression across the array surface. Spottedmicroarray features such as housekeeping genes, sample pools, genomicDNA, or all genes on a microarray are typically used for normalization.Normalization based on these features is not always appropriate,especially for smaller focussed arrays (versus whole genome microarrays)where unbalanced changes are likely to occur, and will have significanteffects on the relative hybridization signal intensities betweenbiological samples. As a result, normalization based on such featureswill give rise to inaccurate interpretations of gene expression data.

According to WO2004/064482, normalization and quality assessment ofmicroarray data, where unbalanced gene expression is anticipated, can beaccomplished by the addition of several different external, non-speciesnucleic acid targets of different concentrations into the RNA sample ofinterest prior to labelling and hybridization. Different concentrationsof external control targets are chosen to mimic a broad range ofexpression profiles. Probes complimentary to the external targets areprinted at equivalent concentrations on the microarray. Variationbetween external control target concentrations in the sample results indifferent fluorescence intensities detected for each external controlprobe. Since detection of the different external controls will beequivalent between RNA samples, and are not affected by unbalanced orglobal shifts in gene expression within the RNA sample of interest, theycan be used for accurate normalization and interpretation of geneexpression data from focussed microarrays.

The drawback to using an external control, where varying amounts ofdifferent targets are added to the RNA sample of interest, is that itrequires accurate measurement of extremely small quantities of thoseseveral RNA targets at low concentrations. The technical errorassociated with measurements at the low range required for microarrayanalysis results in unacceptable variation between samples that willhave a significant influence on normalization and interpretation of geneexpression data. In addition, the optimization and preparation ofmultiple external control targets and probes is time-consuming andcostly.

Accordingly, there is a need for a microarray system that allows formore accuracy in the normalization of the data.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method that satisfiesthe above-mentioned need.

More particularly, the present invention provides a microarray and ahybridizing reagent that allow for more accuracy in the normalization ofdata.

The present invention teaches the addition of at least one externalnon-species nucleic acid or protein target, which can be accuratelymeasured, to each biological sample prior to labelling and hybridizationand the concurrent use of a matched external probe (either DNA, RNA orantibody) printed on the microarray in a series of differentconcentrations. The dilution of a control probe in situ (i.e. printed onthe hybridization surface in different concentrations) effectivelymimics a broad range of expression profiles. This approach allows anexperimenter to apply at least one external control target spiked intoRNA or protein samples reducing the preparation time a cost normallyassociated with the development and use of external controls.

Variation in the amount of printed probe will result in variation of theamount of hybridized external control target resulting in the detectionof a broad range of hybridization signal intensities. Since the amountof external control target added to each biological sample isequivalent, and unaffected by unbalanced or global shifts in expressionwithin the sample, detection of the external control will be equivalentacross all samples. These external control features can then be utilizedfor normalization, quality control, validation or other applications.

The addition of a constant and an accurately measurable quantity ofexternal control target to each biological sample prior to labelling andhybridization, combined with the printing of several differentconcentrations of the external control probe covering the dynamic rangeof signal detection, represents a novel approach to microarraynormalization, quality control, validation and other applications thatis unaffected by unbalanced or global shifts in gene expression.

The present invention presents the advantage of not relying on pipettingdifferent concentrations and volumes of external DNA/RNA into reactionmixtures and is therefore much more accurate.

The present invention also has the advantage of providing means ofcarrying out in situ dilution in microarrays that are less expensive tooperate and are not time consuming. In addition, the present inventionpresents the advantage of providing means of carrying out in situdilution in microarrays used in toxicological studies or in comparingtwo states of a cell or in a study requiring the use of arrays ormicroarrays.

The present invention thus provides for an array system comprising:

a solid support having at least one grid;

at least one external control probe attached to at least one grid of thesolid support in a series of concentrations, the concentrations arerandomized in each grid;

a hybridizing mixture containing at least one external control targetcomplementary to the external control probe; and

a plurality of sample probes attached to the solid support at discretelocations.

The present invention also provides a process of normalizing an arraysystem, comprising the steps of:

a) providing a solid support comprising at least one grid;

b) attaching at least one external control probe to said solid supportin a series of concentrations, each of said series of concentration ofsaid at least one external control probe being attached to a grid ofsaid solid support at a different discrete location from anotherconcentration of said series, said series of concentrations beingrandomized in said grid;

c) attaching at least one sample probe on at least one grid to saidsolid support;

d) making several grids by repeating steps a) and b);

e) hybridizing the hybridizing mixture to the microarray;

f) measuring level of hybridization between the at least one externalcontrol probe and the at least one external control target and betweenthe plurality of sample probes and plurality of sample targets; and

g) determining amount of sample target present in the hybridizingmixture.

The present invention also provides a kit comprising:

a solid support having at least one grid;

at least one external control probe attached to at least one grid ofsaid solid support in a series of concentrations randomized in eachgrid;

a hybridizing mixture containing at least one external control targetcomplementary the external control probe; and

a plurality of sample probes attached to the solid support at discretelocations.

Other objects and advantages of the present invention will be apparentupon reading the following non-restrictive detailed description, madewith reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows a grid layout according to the invention.

FIGS. 2A, B and C show other grid layouts according to the invention.

FIG. 3 shows a diagrammatic representation of microarray printing withVirTek ChipWritePro® (BioRad) using 12 pins.

FIG. 4 shows the ToxArray® genes sorted by ontology.

FIG. 5 is images of identical hybridizations of oligo test set on twoslide types showing (arrows) the increase in detectable test oligo spotson the PowerMatrix® slide (a) compared to the QMT Epoxy® slide (b).

FIG. 6 shows the effect of oligo spotting buffer on mean spot intensity.

FIG. 7 shows a partial microarray image of Hsp84-1 oligo printed in 4different printing buffers on the PowerMatrix® slide.

FIG. 8 shows the effect of oligo printing concentration on signalintensities. A is the logarithmic plot of mean spot intensities of asubset of test oligos printed at different concentrations. B is theimage of a subset of RpL5 oligo printing concentration.

FIG. 9 shows a typical microarray image of oligos test set printed at 40μM, in ArrayIT® spotting solution (Telechem International) on thePowerMatrix® slide (Full Moon Biosystems).

FIG. 10 shows the outline of an externally controlled microarrayexperiment.

FIG. 11 shows the mean signal intensities of external control dilutionsand test oligo features.

FIG. 12 shows the external control dilution series spot intensities. Ashows the logarithmic plot of external dilution series spot intensitiesfor 5 independent microarray experiments (Cy5 and Cy3). B shows a subsetof external control features from a typical microarray image.

FIG. 13 shows signal intensities for a single external control dilutionsorted by array grid position.

FIG. 14 shows signal intensities for a single external control dilutionsorted by printing pin.

FIG. 15 shows a linear plot of external control dilution series.

FIG. 16 shows replicate external control spots sorted by grid andmetacolumn.

LIST OF TABLES

Table 1 is a list of positive control house keeping genes.

Table 2 is a list of the commercial glass slides tested.

Table 3 is a list of the oligonucleotides printing buffers tested.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Definitions:

In order to provide an even clearer and more consistent understanding ofthe description, including the scope given herein to such terms, thefollowing definitions are provided:

Gene refers to nucleic acid sequences (including both RNA or DNA) thatencode genetic information for the synthesis of a whole RNA, a wholeprotein, or any portion of such whole RNA or whole protein.

Probe refers to a nucleotide sequence often an oligonucleotide that is,or is intended to be, attached to a solid support in an array that canbe used to hybridize with and thereby identify the presence of acomplementary sequence, or a complementary sequence differing from theprobe sequence but not to a degree that prevents hybridization under thehybridization stringency conditions used. As is well known to thoseskilled in the art, for the hybridization characteristics to be similaracross a wide range of oligonucleotides, it is typically required thatthe probes on the array be of the substantially same length, have asimilar percentage of Guanine to Cytosine content and lack any extensiveruns of poly A, poly G, poly C, or poly T tracts. The goal ofcontrolling these parameters is to produce probes that have similarmelting and hybridization temperatures. Additionally, these probesshould, preferably, lack length complementary regions and not formhairpin structures. The probe can be DNA, RNA, oligonucleotides, cDNA,proteins or antibodies.

Target refers to nucleic acids intended to be hybridized (or bound) toprobes immobilized on microarrays by sequence complementarity. Thetarget can be DNA, RNA, oligonucleotides, cDNA, proteins or antibodies.As is well-known in the art, target nucleic acids may be obtained from awide variety of organisms (bacteria, plants . . . ), tissues or cells.Methods and techniques for the extraction, manipulation and preparationof nucleic acids for hybridization reactions are well-known in the art(see, for example, J. Sambrook et al., “Molecular Cloning: A LaboratoryManual”, 1989, 2nd Ed., Cold Spring Harbor Laboratory Press: New York,N.Y.; “PCR Protocols: A Guide to Methods and Applications”, 1990, M. A.Innis (Ed.), Academic Press: New York, N.Y.; P. Tijssen “Hybridizationwith Nucleic Acid Probes-Laboratory Techniques in Biochemistry andMolecular Biology (Parts I and II)”, 1993, Elsevier Science; “PCRStrategies”, 1995, M. A. Munis (Ed.), Academic Press: New York, N.Y.;and “67 Protocols in Molecular Biology”, 2002, F. M. Ausubel (Ed.), 5thEd., John Wiley & Sons).

Complement when used in reference to a given polynucleotide sequencerefers to a sequence of nucleotides which can form a double-strandedheteroduplex in which every nucleotide in the sequence of nucleotides isbase-paired by hydrogen bonding to a nucleotide opposite it in theheteroduplex with the given polynucleotide sequence. The term may referto a DNA or an RNA sequence that is the complement of another RNA or DNAsequence. As used herein, the term “hybridizes” refers to the formationof a hydrogen-bonded heteroduplex between two nucleic acid molecules.Generally, a given nucleic acid molecule will hybridize with itscomplement, or with a molecule that is sufficiently complementary to thegiven molecule to permit formation of a hydrogen-bonded heteroduplexbetween the two molecules.

Oligonucleotide Oligonucleotides means nucleic acid, eitherdesoxyribonucleic acid (DNA), or ribonucleic acid (RNA), insingle-stranded or double-stranded form and having one nucleotide ormore whether, occurring naturally or non-naturally in a particular cell,tissue or organism, and any chemical modifications thereof. Suchmodifications include, but are not limited to providing other chemicalgroups that incorporate additional charge, polarizability, hydrogenbonding or electrostatic interaction to one or more of nucleic acidbases of the oligonucleotide.

Specifically hybridizing refers to the association between twosingle-stranded nucleotide molecules of sufficiently complementarysequence to permit such hybridization under pre-determined conditionsgenerally used in the art (sometimes termed “substantiallycomplementary”). In particular, the term refers to hybridization of anoligonucleotide with a substantially complementary sequence containedwithin a single-stranded DNA or RNA molecule of the invention, to thesubstantial exclusion of hybridization of the oligonucleotide withsingle-stranded nucleic acids of non-complementary sequence.

Toxicant refers to any substance potentially toxic. It also refers achemical having the qualities or effects of a poison and to a harmfulsubstance or agent that may injure an exposed organism. Toxicantsaccording to the present invention may be chemical toxicants like forinstance cyanides, phenols, pesticides, or heavy metals. Toxicants mayalso be physical in nature like for instance asbestos, silicas and theymay also be radioactive.

Normalization refers to the process of removing superfluous differencesin array or microarray data by reducing them to a common denominator.

External refers to a molecule that does not hybridize with the moleculesof the collection or sample under study. In a preferred embodiment, anucleic acid sequence is thus “external” if its complement is notpresent in the nucleic acids of a collection. The source or collectionmay preferably be a plurality of nucleic acids to be hybridized to anarray. The external control probe has a sequence that does not hybridizeto a target from the plurality of targets from an organism under studyto be hybridized on an array or microarray.

Hybridizing mixture refers to a mixture being or intended to behybridized to an array. Those of ordinary skill in the art willappreciate that the hybridizing sample may contain DNA, RNA, or both,but most commonly contains cDNA. The hybridizing mixture typicallycontains nucleic acids whose hybridization with probes on an array isdetectable. For example, in many embodiments, the hybridizing mixturecomprises or consists of detectably labelled nucleic acids.

Labelled, Detectably labelled, labelled with a detectable agent: specifythat a nucleic acid molecule or individual nucleic acid segments from asample can be detected and/or visualized following binding (i.e.hybridization) to probes immobilized on an array. The detectable agentis such that it generates a signal which can be measured and whoseintensity is related to the amount of hybridized nucleic acids. Thedetectable agent is such that it generates a localized signal, therebyallowing spatial resolution of the signal from each spot on the array.Methods for labelling nucleic acid molecules are well known in the art.Suitable detectable agents include, but are not limited to: variousligands, radionuclides, fluorescent dyes, chemiluminescent agents,microparticles, enzymes, calorimetric labels, magnetic labels, andhaptens.

Fluorescent label refers to a molecule, which, in solution and uponexcitation with light of appropriate wavelength, emits light back.Numerous fluorescent dyes of a wide variety of structures andcharacteristics are suitable for use in the practice of this invention.Similarly, methods and materials are known in the art for fluorescentlylabelling nucleic acids (see, for example, R. P. Haugland, “MolecularProbes: Handbook of Fluorescent Probes and Research Chemicals1992-1994”, Sti' Ed., 1994, Molecular Probes, Inc.). In choosing afluorophore, it is generally preferred that the fluorescent moleculeabsorbs light and emits fluorescence with high efficiency (i.e., it hasa high molar absorption coefficient and a high fluorescence quantumyield, respectively), and is photostable (i.e. it does not undergosignificant degradation upon light excitation within the time necessaryto perform the array-based hybridization). Suitable fluorescent labelsfor use in the practice of the methods of the invention include, forexample, Cy-3, Cy-5, Texas red, FITC, Spectrum Red, Spectrum Green,Alexa-488, phycoerythrin, rhodamine, fluorescein, fluoresceinisothiocyanine, carbocyanine, merocyanine, styryl dye, oxonol dye,BODIPY dye, and equivalents, analogues or derivatives of thesemolecules.

Array refers to an arrangement on a solid support of multiple nucleicacid molecules of known or unknown sequences. These nucleic acidmolecules are attached to discrete “spots” or positions on the support.A discrete spot may contain a single nucleic acid molecule or a mixtureof different nucleic acid molecules. Spots on an array may be arrangedon the support surface at different densities. In general, microarrayswith probe pitch smaller than 500 pm (i.e., density larger than 400probes per cm²) are referred to as high density microarrays, otherwise,they are called low density microarrays. Arrays come as two-dimensionalprobe matrices (or supports), which can be solid or porous, planar ornon-planar, unitary or distributed. The term “microarray” morespecifically refers to an array that is miniaturized so as to requiremicroscopic examination for visual evaluation. Arrays used in theinvention are preferably microarrays.

Expression refers to the process by which nucleic acid is transcribedinto mRNA and translated into peptides, polypeptides, or proteins.

Nucleic acid refers to either desoxyribonucleic acid (DNA), orribonucleic acid (RNA).

Polynucleotide refers to any DNA, RNA sequence or molecule having onenucleotide or more, including nucleotide sequences encoding a completegene. The term is intended to encompass all nucleic acids whetheroccurring naturally or non-naturally in a particular cell, tissue ororganism. This includes DNA and fragments thereof, RNA and fragmentsthereof, cDNAs and fragments thereof, expressed sequence tags,artificial sequences including randomized artificial sequences.

Kit may comprise packages, each containing one or more of the variousreagents (typically in concentrated form) required to perform therespective tests. The kits according to these embodiments of theinvention are contemplated to be useful for detecting and/or quantifyingDNA, RNA, and/or protein in biological (or other types of) samples.

General Overview of the Invention

The present inventors have discovered that it is possible to normalizemicroarrays by using external control probes in a series of dilutionsacross the microarray itself. Therefore the present invention isdirected towards an array or a microarray having a series of dilutionsof an external probe attached to it, a process of normalization by usingsuch a series of concentrations of an external probe and also a kitcomprising the array containing the series of concentrations of theexternal probe and a hybridizing buffer containing the external controltarget.

Other objects and advantages of the present invention will be apparentupon reading the following non-restrictive description of severalpreferred embodiments and the accompanying examples.

Description of Preferred Embodiments

The inventors have found a way to normalize an array or a microarray byusing a series of concentrations of an external control probe spanningall areas of a grid in the array or microarray. Thus the presentinvention provides for an array system comprising:

a solid support having at least one grid;

at least one external control probe attached to the grid in a series ofconcentrations which is randomized in the grid;

a hybridizing mixture containing at least one external control targetcomplementary to the external control probe; and

a plurality of sample probes attached to the solid support at discretelocations.

According to a preferred embodiment of the invention, the array systemis a microarray system.

In a preferred embodiment, the sample probes and the external controlprobes are printed on the solid support. In a more preferred embodimentthe printing is done by a Virtek ChipWriterPro® (BioRad). According toanother preferred embodiment of the invention, the Virtek ChipWriterPro®has 12 pins, and as can be seen in FIG. 1, each pin produces a distinctgrid with x rows and y columns. The external control probe is a moleculethat does not hybridize with the molecules of the collection or sampleunder study. This in situ approach of printing of the series ofconcentrations of the external control probe, does not rely on pipettingdifferent concentrations and volumes of different RNA/DNA into reactionmixture and is therefore much more accurate.

The array system according to the invention is further characterized bya hybridizing mixture, which comprises a plurality of sample targets tobe hybridized on the array. According to a preferred embodiment of theinvention, the external control probe and the sample probes of the arrayare chosen from DNA, RNA, oligonucleotides, cDNA, proteins andantibodies and more preferably they are oligonucleotides, and even morepreferably 70mer oligonucleotides. The external control probe may befrom a bacterial, plant, animal gene or from any other organism. In apreferred embodiment of the invention, the external control probe is anoligo probe designed for the Arabidopsis thaliana chlorophyll synthasegene and the external control target is RNA prepared through in vitrotranscription of A. thaliana chlorophyll synthase cDNA.

The concentration of the external control probe is chosen to start atextremely low molarity and going up to saturation. These concentrationsare chosen to mimic the range of the expression profile of the studiedsample and allow the control for differential hybridization occurringacross the chip. In a preferred embodiment of the invention, the seriesof concentrations of the external control probe varies from 0.0015 to100 μM. In a more preferred embodiment of the invention, the series ofconcentrations of the external control probe is 0.0015, 0.0031, 0.0061,0.0122, 0.0244, 0.0488, 0.0077, 0.195, 0.39, 0.78, 1.5625, 3.125, 6.25,12.5, 25, 50, 60, 80 and 100 μM. As can be appreciated from FIGS. 2A and2B, showing preferred embodiments of the invention, the series ofconcentrations is spatially randomized and duplicated on a grid. A gridalso contains at least one spot with buffer, spots that are left emptyand at least one spot printed with a housekeeping gene, the housekeeping gene probe being preferably RpL5.

As mentioned above, the sample probes are chosen from DNA, RNA,oligonucleotides, cDNA, proteins and antibodies. These samples probesare representative of relevant genes under study. In a preferredembodiment, the relevant genes under study are toxicologically relevantgenes. According to a preferred embodiment, the sample probes areoligonucleotides. In another preferred embodiment of the invention thesample probes oligonucleotides are 70mer oligonucleotides. In yetanother preferred embodiment of the invention, the 70meroligonucleotides are designed and synthesized according to the followingcharacteristics:

less than 60% G C content

less than 1000 basis from the 3′ end;

longest stem being less than 9 bases;

less than 20% cross homology; and/or

less than 20 contiguous bases in common with top BLAST hit gene.

The invention is further characterized in that the sample probes areattached to the solid support in a fixed concentration. Hence, the 70meroligonucleotides of the invention are attached to the solid support in afixed concentration. In a preferred embodiment of the invention, the70mer oligonucleotides are attached to the solid support at aconcentration chosen from 60, 40, 30, 15, 7.5 and 3.5 μM and preferablyat the concentration of 40 μM. In a preferred embodiment of theinvention, the 70mer oligonucleotides are representative oftoxicologically relevant genes in regulatory toxicology and riskassessment studies.

As can be appreciated in FIG. 1, according to a preferred embodiment ofthe invention, the array system is further characterized in that theseries of concentrations is spatially randomized across a grid andattached in duplicate in each grid of the array. Having at least twocopies of different dilutions of the external control spanning the areaacross a chip, allows the control of differential hybridization. As canbe appreciated also from FIG. 1:

The array system is further characterized in that the series ofconcentrations of the external control probe varies from 0.0015 to 100μM. More preferably the series of concentrations of the at least oneexternal control probe is 0.0015, 0.0031, 0.0061, 0.0122, 0.0244,0.0488, 0.0077, 0.195, 0.39, 0.78, 1.5625, 3.125, 6.25, 12.5, 25, 50,60, 80 and 100 μM.

The invention also provides for the process of normalizing an arraysystem, comprising the steps of:

-   -   a) providing a solid support comprising at least one grid;    -   b) attaching at least one external control probe to the solid        support in a series of concentrations, each concentration of the        series is attached to a grid of the array at a different        discrete location, the concentrations are also randomized in the        grid;    -   c) attaching sample probes spatially arranged in the grid;    -   d) making several grids on the solid support by repeating        steps a) and b);    -   e) hybridizing the hybridizing mixture to the microarray;    -   f) measuring level of hybridization between the at least one        external control probe and the at least one external control        target and between the plurality of sample probes and plurality        of sample targets; and    -   g) determining amount of sample target present in the        hybridizing mixture.

In a preferred embodiment of the invention the series of concentrationof the external control probe of the process varies from 0.0015 to 100μM and even more preferably it is 0.0015, 0.0031, 0.0061, 0.0122,0.0244, 0.0488, 0.0077, 0.195, 0.39, 0.78, 1.5625, 3.125, 6.25, 12.5,25, 50, 60, 80 and 100 μM.

In another preferred embodiment of the invention the sample probes andthe external control probes are from DNA, RNA, oligonucleotides, cDNA,proteins and antibodies, more preferably oligonucleotides. In apreferred embodiment of the invention the sample probes of the processare 70mer oligonucleotides, more preferably designed and according tothe following characteristics:

less than 60% G C content

less than 1000 basis from the 3′ end;

longest stem being less than 9 bases;

less than 20% cross homology; and/or

less than 20 contiguous bases in common with top BLAST hit gene.

According to a preferred embodiment of the invention, the process isfurther characterized in that it comprises the following step:

-   -   h) attaching the at least one external control probe series of        concentration in duplicate in the grid.

In another preferred embodiment of the invention, the process is furthercharacterized in that it comprises at least one of the following steps:

-   -   i) adding a buffer only to at least one discrete spot on the        grid;    -   j) leaving at least one discrete spot in said grid empty;    -   k) attaching a random hexamer to one spot on said grid;    -   l) attaching a random pool of 70mer oligonucleotides to at least        one discrete spot on said grid; and    -   m) attaching a probe from a housekeeping gene to at least one        discrete spot on said grid.

In a preferred embodiment of the invention, the 70mer oligonucleotidesare attached to the solid support at a concentration chosen from 60, 40,30, 15, 7.5 and 3.5 μM and more preferably at 40 μM.

The composition and the method of preparation of the buffer of step e)is well known in the art. In a preferred embodiment of the presentinvention, the buffer is PBS®, Quantifoll I®, Quantifoll II® andArrayIT®, and more preferably ArrayIT®. In a preferred embodiment of theinvention, the housekeeping gene is RpL5.

In a preferred embodiment of the invention, and as can be seen fromFIGS. 2A, 2B, and 2C, the buffer can be added to one spot, 6 or 7 spotsin the grid. Also in a preferred embodiment of the invention, no spot orone or 4 spots of the grid are left empty. In a preferred embodiment,the house keeping gene is RpL5. Also in a preferred embodiment of theinvention, the housekeeping gene probe is attached to one, 3 or 4 spotsin the grid, and a random pool of 70mer oligonucleotides is attached to3 spots in the grid. The series of concentrations of external controlprobe is also duplicated across the grid spanning all its area. In amore preferred embodiment, the housekeeping gene is RpL5.

The process of the invention is even further characterized in that itcomprises the step of:

-   -   n) attaching a plurality of sample probes in other discrete        spots of each said grid as can also be appreciated from FIGS.        2A, 2B and 2C.

The process of the invention is further characterized by that thehybridization mixture comprises a plurality of sample targets to behybridized on the array. The hybridization, the measurement of the levelof hybridization and the determination of the amount of sample targetpresent in the hybridizing mixture are done according to methods wellknown by a person in the art.

According to a preferred embodiment of the invention, and as can be seenin FIG. 3, the external control probe and the sample probes are printedon the solid support, and more preferably by a Virtek ChipWriterPro®(BioRad). According to another preferred embodiment of the invention,the Virtek ChipWriterPro® has 12 pins, and as can be seen in FIG. 1,each pin produces a distinct grid with x rows and y columns. In apreferred embodiment of the invention, each grid can be 12×12, 12×13 or12×14. The 12 distinct grids printed by each pin represent a supergridcluster. The supergrid cluster is then replicated across the surface ofthe glass slide to produce the final microarray. In a preferredembodiment of the invention, the supergrid is replicated 4 times as canbe appreciated in FIGS. 1 and 2C.

The present invention also provides for a kit comprising:

a solid support having at least one grid;

at least one external control probe, said at least one external controlprobe being attached to at least one grid of said solid support in aseries of concentrations, said series of concentrations being randomizedin said at least one grid;

a hybridizing mixture containing at least one external control targetbeing complementary to said at least one external control probe; and

a plurality of sample probes attached to said solid support at discretelocations.

The kit is further characterized in that the hybridizing mixture furthercomprises a plurality of sample targets to be hybridized on the array.

In a preferred embodiment of the invention the series of concentrationsof the external control probe of the kit varies from 0.0015 to 100 μM.and more preferably it is 0.0015, 0.0031, 0.0061, 0.0122, 0.0244,0.0488, 0.0077, 0.195, 0.39, 0.78, 1.5625, 3.125, 6.25, 12.5, 25, 50,60, 80 and 100 μM.

In a preferred embodiment of the invention the external control probeand the sample probes of the kit of the invention are chosen from DNA,RNA, oligonucleotides, cDNA, proteins and antibodies and more preferablythe external control probe and the sample probes of the kit areoligonucleotides, the oligonucleotides are even more preferably 70meroligonucleotides. In a preferred embodiment of the invention 70meroligonucleotides of the kit are designed and synthesized according tothe following characteristics:

less than 60% G C content

less than 1000 basis from the 3′ end;

longest stem being less than 9 bases;

less than 20% cross homology; and/or

less than 20 contiguous bases in common with top BLAST hit gene.

In another preferred embodiment of the invention the 70meroligonucleotides of the kit are attached to the solid support at aconcentration chosen from 60, 40, 30, 15, 7.5 and 3.5 μM and morepreferably at 40 μM.

EXAMPLES

The following examples are illustrative of the applicability of thepresent invention and are not intended to limit its scope. Modificationsand variations can be made therein without departing from the spirit andscope of the invention. Although any method and material similar orequivalent to those described herein can be used in the practice fortesting of the present invention, the preferred methods and materialsare described. The following experimental procedures and materials wereused for the examples set fort below in the normalization of theinventors ToxArray®.

A. Materials and Methods

1. Gene Selection and Probe Design

Genes predictive of toxicant exposure were established from in housegene expression data, gene expression studies in the current literature,and statistical re-analysis of publicly available microarray data.

The following information was acquired through publicly availabledatabases for each gene:

Official gene symbol and name

Gene sequence (RefSeq or GenBank accession number)

UniGene Cluster ID

Alternate gene names and aliases

Ontology

LocusLink ID

Ensemble Gene ID

Ensembl Transcript ID

The current list consists of 1100 genes. FIG. 4 displays the gene listgrouped by ontology.

Custom 70mer oligonucleotides were designed and synthesized for eachgene by Qiagen according to the following criteria:

less than 60% GC content

less than 1000 bases from the 3′ end

Longest stem less than 9 bases

less than 70% cross homology

less than 20 contiguous bases in common with top BLAST hit gene.

2. Oligonucleotide Test Set

Prior to printing the high density array, a set of control oligosprovided by Qiagen were used to optimize array printing andhybridization protocols. These comprise:

-   -   Positive control oligos designed for common housekeeping genes        (Table 1).

Random 70mer oligos designed to have no cross homology to any knownmouse genes. TABLE 1 Gene Symbol Gene Name Ldh1 Lactate dehydrogenase 1,A chain Hsp70-3 Heat shock protein, 70 kDa 3 GapdGlyceraldehyde-3-phosphate dehydrogenase Ubb Ubiquitin B Tcea1Transcription elongation factor A (SII) 1 Eif4a2 Eukaryotic translationinitiation factor 4A2 Cyc1 Cytochrome c-1 Tcfe2a Transcription factorE2a Nup62 Nucleoporin 62 Rpl5 Ribosomal protein L5 Top1 Topoisomerase(DNA) I Hsp84-1 Heat shock protein, 84 kDa 1

In addition to the test oligos provided by Qiagen, buffer-only andrandom hexamers were also printed on our initial microarrays as negativecontrol features.

3. In House Microarray Printing

Microarray printing was performed by the Virtek ChipWriterPro (BioRad).An example of printing a microarray using 12 printing pins isrepresented in FIG. 3.

4. Target Labeling and Hybridization Protocols

Universal mouse reference RNA (Stratagene) was used for all targetlabelling and array hybridizations. All array experiments were carriedout in two colours, Cy5 and Cy3, using the cRNA linear amplification andfluorescent labelling kit from Agilent Technologies. All arrays wereprocessed in an automated hybridization station (Tecan), images wereobtained with ScanArray Express® confocal laser scanner (PackardBioSystems), and image analysis was performed by QuantArray® imageanalysis software (Packard Biosystems).

B. Results

1. Glass Slide and Oligo Printing Buffer Selection

The PowerMatrix® and QMT Epoxy® slides produced significantly lowerbackground signals compared to all other slides tested (data not shown).The PowerMatrix® slide was chosen over the QMT Epoxy® slide because ofits ability to detect more of the test oligo spots (FIG. 5).

Oligo spotting buffer had a significant effect on spot intensity:

ArrayIT® spotting solution produced spots with the highest signalintensities while PBS and Quantifoil I® spotting solutions producedspots with the lowest signal intensities (FIG. 6). ArraylT® alsoproduces the best quality spots based on spot size and morphology (FIG.7).

Tables 2 and 3 list respectively the glass slides and the oligo printingbuffers tested. TABLE 2 Slide Name Manufacturer QMT Epoxy QuantifoilMicro Tools GmbH PowerMatrix Full Moon Biosystems SuperChip epoxysilaneErie Scientific SuperAldehyde Telechem International CodeLink ActivatedAmersham Biosciences SpotOn Scandinavian Micro Biodevices

TABLE 3 Oligo Printing Buffer Manufacturer ArraylT TelechemInternational Spotting Solution I Quantifoil Micro Tools GmbH SpottingSolution II Quantifoil Micro Tools GmbH Phosphate Buffered Saline (PBS)In house

2. Optimization of Oligo Printing Concentration

To determine the optimal printing concentration, the oligo test set wasprinted at 60, 40, 30, 15, 7.5, and 3.75 μM.

Large intensity gains are apparent when the oligo printing concentrationis increased to 40 μM (FIG. 8).

Intensity gains were minimal for printing concentrations >40 μM (FIG.8).

The optimal oligo printing concentration was determined to be 40 μM,FIG. 9 displays a typical microarray image of the oligo test set printedunder optimal oligo printing concentration, printing buffer, and glassslide.

3. Microarray Normalization Feature

The majority of microarray analyses rely on normalization methods thatassume evenly distributed changes and/or absence of global shifts usingmicroarray features such as housekeeping genes, spotted microarraysample pools, genomic DNA, or all genes.

Normalization based on the above features are not appropriate for theinventors ToxArray® because global unbalanced changes are expected dueto its relatively small size and the fact that genes were chosen basedon evidence of transcript level changes as a result of toxicantexposure.

The use of external control features, made up of a non-murineoligonucleotide and specific RNA, allows a better comparison betweenslides and overcomes the problem of global changes in gene expression.

In addition to their use in normalization, external control features canalso be used to cope with local, intensity-dependent systematicvariation by representation in sufficient numbers in each grid and tomonitor sample labelling and optimization of microarray hybridizationprotocols.

4. External Control Feature Design

The inventors have opted to add a constant quantity of external RNA toeach total RNA sample while varying the amounts of external controloligo printed on the array (FIG. 10).

An oligo probe was designed for the Arabidopsis thaliana chlorophyllsynthase gene.

External control target RNA was prepared through in vitro transcriptionof A. thaliana chlorophyll synthase cDNA present in a plasmid providedby the Ontario Cancer Institute. The chlorophyll synthase target RNA isadded into the mouse target RNA, labeled with a fluorescent dye, andhybridized to the array. All methods followed are methods known in theart.

External control features of different concentrations will produce spotswith different intensities as a result of hybridization of the externaltarget RNA to the oligo content in the spot.

5. Optimization of External Control Oligo Dilution Series

Microarrays were printed with external control oligo dilutions rangingfrom 100 μM to 0.0015 μM. External control RNA was spiked into referenceRNA (Stratagene) prior to labelling. 2 ng, 8 ng, 18 ng, 20 ng, and 40 ngexternal control RNA spikes were tested. A series of 19 external controldilutions was found to be optimal, covering the complete range ofexpression levels observed for the test oligo set. (FIG. 11) andreproducible across experiments (FIG. 12).

6. Microarray Feature Printing Design

Systematic errors arising reproducibly as a result of experimentalprocedure contribute to inaccuracies in measured gene expression levels.

The spatial arrangement of features on a microarray resulting fromprinting design is one of the major sources of systematic error:

In FIG. 13 the spatial periodicity of spot intensity based on array gridposition is evident.

When the same data is sorted by printing pin (FIG. 14), a cleardifference in spot intensity for the same feature is observed based onprinting pin.

The ToxArray® has been designed such that all control and externalnormalization features are printed by each pin resulting in theirpresence in every grid on the array.

Each grid on the ToxArray® is printed in quadruplicate over the arrayarea to account for within-slide spatial variations.

Each grid consists of 96 unique gene features and an identical layout ofcontrol features (FIG. 1) including:

external control dilutions

RpL5 housekeeping gene

negative controls (buffer-only and randomized 70mers).

7. Quality Test

As can be appreciated from FIG. 2, by starting at extremely low molarityand going up to saturation, the inventors span the linear dynamic rangeof the signal intensities, allowing the choice of one or several spotsto use within each subgrid for normalizing.

FIG. 15 shows the result from a recent quality test of the inventorsTOXARRAY® plotting an external control dilution series, ensuring thatwithin a subgrid one is spanning the range of signal intensities frombackground to saturation. This allows to choose an appropriate spot ortwo within each subgrid to normalize grids from top to bottom. As can beappreciated from FIG. 16, the system can thus can be used to simplydetect differential hybridization (increasing grid number is going downthe chip—if the line is sloping down or up, it indicates signalintensity of the same spot differs from top to bottom of the chip).

The inventors have thus designed an oligonucleotide-based microarrayrepresenting more than 1100 genes predictive of toxicant exposure basedon gene expression data in current literature, in house data, andstatistical re-analysis of relevant existing microarray data in which anexternal control feature for normalization has been designed and testedfor its ability to mimic the range of observed expression levels for atest set of oligos. The individual grid layout and number of control andexternal normalization features per grid have been designed to cope withsources of both systematic error and spatial variation.

1. An array system comprising: a solid support having at least one grid;at least one external control probe, said at least one external controlprobe being attached to at least one grid of said solid support in aseries of concentrations, said series of concentrations being randomizedin said at least one grid; a hybridizing mixture containing at least oneexternal control target being complementary to said at least oneexternal control probe; and a plurality of sample probes attached tosaid solid support at discrete locations.
 2. The array system accordingto claim 1, wherein the hybridizing mixture further comprises aplurality of sample targets to be hybridized on said array.
 3. The arraysystem according to claim 1, wherein the at least one external controlprobe and the sample probes are selected from the group consisting ofDNA, RNA, oligonucleotides, cDNA, proteins and antibodies.
 4. The arraysystem according to claim 1, wherein said series of concentrations ofthe at least one external control probe varies from 0.0015 to 100 μM. 5.The array system according to claim 4, wherein said series ofconcentrations of the at least one external control probe is 0.0015,0.0031, 0.0061, 0.0122, 0.0244, 0.0488, 0.0077, 0.195, 0.39, 0.78,1.5625, 3.125, 6.25, 12.5, 25, 50, 60, 80 and 100 μM.
 6. The arraysystem according to claim 1, wherein the sample probes areoligonucleotides.
 7. The array system according to claim 6, wherein theoligonucleotides are 70mer oligonucleotides.
 8. The array systemaccording to claim 7, wherein the 70mer oligonucleotides are designedand synthesized according to the following characteristics: less than60% G C content less than 1000 bases from the 3′ end; longest stem beingless than 9 bases; less than 20% cross homology; and/or less than 20contiguous bases in common with top BLAST hit gene.
 9. The array systemaccording to claim 8, wherein the 70mer oligonucleotides are attached tothe solid support at a concentration chosen from 60, 40, 30, 15, 7.5 and3.5 μM.
 10. The array system according to claim 9, wherein the 70meroligonucleotides are attached to the solid support at a concentration of40 μM.
 11. The array system according to claim 1, wherein it is amicroarray system.
 12. The array system according to claim 1, whereinsaid at least one external control probe and said plurality of sampleprobes are printed on said solid support.
 13. A process of normalizingan array system, comprising the steps of: a) providing a solid supportcomprising at least one grid; b) attaching at least one external controlprobe to said solid support in a series of concentrations, each of saidseries of concentration of said at least one external control probebeing attached to a grid of said solid support at a different discretelocation from another concentration of said series, said series ofconcentrations being randomized in said grid; c) attaching at least onesample probe on at least one grid to said solid support; d) makingseveral grids by repeating steps a) and b); e) hybridizing thehybridizing mixture to the microarray; f) measuring level ofhybridization between the at least one external control probe and the atleast one external control target and between the plurality of sampleprobes and plurality of sample targets; and g) determining amount ofsample target present in the hybridizing mixture.
 14. The processaccording to claim 13, further comprising the following step: h)attaching the at least one external control probe series ofconcentration in duplicate on said grid.
 15. The process according toclaim 13, further comprising at least one of the following steps: i)adding a buffer only to at least a discrete spot on said grid; j)leaving at least one discrete spot in said grid empty; k) attaching arandom hexamer to one spot on said grid; l) attaching a random pool of70mer oligonucleotides to at least one discrete spot on said grid; andm) attaching a probe from a house keeping gene to at least one discretespot on said grid.
 16. The process according to claim 15, wherein thehouse keeping gene is RpL5.
 17. The process of normalization accordingto claim 15, further comprising the step of: n) attaching a plurality ofsample probes in other discrete spots of each said grid.
 18. The processof normalization according to claim 15, wherein the hybridizationmixture comprises a plurality of sample targets to be hybridized on thearray.
 19. The process of normalization according to claim 18, whereinthe at least one external control probe and of the sample probes arechosen from DNA, RNA, oligonucleotides, cDNA, proteins and antibodies.20. The process of normalization according to claim 19, wherein saidseries of concentrations of the at least one external control probevaries from 0.0015 to 100 μM.
 21. The process of normalization accordingto claim 20, wherein said series of concentrations of the at least oneexternal control probe is 0.0015, 0.0031, 0.0061, 0.0122, 0.0244,0.0488, 0.0077, 0.195, 0.39, 0.78, 1.5625, 3.125, 6.25, 12.5, 25, 50,60, 80 and 100 μM.
 22. The process of normalization according to claim21, wherein the sample probes are oligonucleotides.
 23. The process ofnormalization according to claim 22, wherein the oligonucleotides are70mer oligonucleotides.
 24. The process of normalization according toclaim 23, wherein the 70mer oligonucleotides are designed andsynthesized according to the following characteristics: less than 60% GC content less than 1000 basis from the 3′ end; longest stem being lessthan 9 bases; less than 20% cross homology; and/or less than 20contiguous bases in common with top BLAST hit gene.
 25. The process ofnormalization according to claim 24, wherein the 70mer oligonucleotidesare attached to the solid support at a concentration selected from 60,40, 30, 15, 7.5 and 3.5 μM.
 26. The process of normalization accordingto claim 25, wherein the 70mer oligonucleotides are attached to thesolid support at a concentration of 40 μM.
 27. A kit, comprising: asolid support having at least one grid; at least one external controlprobe, said at least one external control probe being attached to atleast one grid of said solid support in a series of concentrations, saidseries of concentrations being randomized in said at least one grid; ahybridizing mixture containing at least one external control targetbeing complementary to said at least one external control probe; and aplurality of sample probes attached to said solid support at discretelocations.
 28. The kit according to claim 27, wherein the hybridizingmixture further comprises a plurality of sample targets to be hybridizedon said array.
 29. The kit according to claim 28, wherein the at leastone external control probe and the sample probes are selected from thegroup consisting of DNA, RNA, oligonucleotides, cDNA, proteins andantibodies.
 30. The kit according to claim 29, wherein said series ofconcentrations of the at least one external control probe varies from0.0015 to 100 μM.
 31. The kit according to claim 30, wherein said seriesof concentrations of the at least one external control probe is 0.0015,0.0031, 0.0061, 0.0122, 0.0244, 0.0488, 0.0077, 0.195, 0.39, 0.78,1.5625, 3.125, 6.25, 12.5, 25, 50, 60, 80 and 100 μM.
 32. The kitaccording to claim 31, wherein the sample probes are oligonucleotides.33. The kit according to claim 32, wherein the oligonucleotides are70mer oligonucleotides.
 34. The kit according to claim 33, wherein the70mer oligonucleotides are designed and synthesized according to thefollowing characteristics: less than 60% G C content less than 1000basis from the 3′ end; longest stem being less than 9 bases; less than20% cross homology; and/or less than 20 contiguous bases in common withtop BLAST hit gene.
 35. The kit according to claim 33, wherein the 70meroligonucleotides are attached to the solid support at a concentrationselected from 60, 40, 30, 15, 7.5 and 3.5 μM.
 36. The kit according toclaim 35, wherein the 70mer oligonucleotides are attached to the solidsupport at a concentration of 40 μM.